Overexpression of tyrosine phosphorylated proteins in reproductive tissues of polycystic ovary syndrome rats induced by letrozole

Objective:To identify the alteration of tyrosine phosphorylated protein expression in rats with polycystic ovary syndrome(PCOS).Methods:Sixteen female Sprague-Dawley rats were divided into the control and letrozole-induced PCOS groups.The oestrus cycle of rats was performed by vaginal smear.Sex hormones and morphology of the ovary,oviduct,and uterus were observed.Expressions and intensity of androgen receptor and tyrosine phosphorylated proteins of reproductive organs were investigated by Western blot.Results:Various polycysts and increased androgen receptor expression were present in the ovary of the PCOS group.The levels of follicle-stimulating hormone and testosteone were significantly higher in the PCOS group while progesterone and estradiol levels were significantly decreased as compared with the control group(P<0.05).Only the size of uterus in the PCOS group was significantly smaller than the control group.However,the density of collagen fibers observed in PCOS uterus was greater than the control group.Moreover,tyrosine phosphorylated proteins were significantly overexpressed in ovary(52,42,and 28 kDa),oviduct(72,56,42,and 28 kDa),and uterus(53 and 42 kDa)of the PCOS group compared to the control group.Conclusions:Presence of tyrosine phosphorylated proteins in the ovary,oviduct and uterus suggests that overexpression of tyrosine phosphorylated proteins may be involved in potential mechanism of female infertility especially in PCOS.

Antioxidant activity and protective effect of Clitoria ternatea flower extract on testicular damage induced by ketoconazole in rats

Background： Ketoconazole （KET）, an antifungal drug, has adverse effects on the male reproductive system. Pre-treatments with antioxidant plant against testicular damage induced by KET are required. The flowers of Clitoria tematea （CT） are proven to have hepatoprotective potential. However, the protective effect on KET-induced testicular damage has not been reported. Objective： To investigate the protective effect of CT flower extracts with antioxidant activity on male reproductive parameters including sperm concentration, serum testosterone level, histopathology of the testis, and testicular tyrosine phosphorylation levels in rats induced with KET. Methods： The antioxidant activity of CT flower extracts was determined using 2,2-diphenyl-1-picrylhydrazyl （DPPH） and ferric reducing antioxidant power （FRAP） assays. Male rats were treated with CT flower extracts （10, 50, or 100 mg/kg BW） or distilled water via a gastric tube for 28 d （preventive period： Days 1-21） and induced by KET （100 mg/kg BW） via intraperitoneal injection for 7 d （induction period： Days 22-28）. After the experiment, all animals were examined for the weights of the testis, epididymis plus vas deferens and seminal vesicle, serum testosterone levels, sperm concentration, histological structures and diameter of testis, and testicular tyrosine phosphorylation levels by immunoblotting. Results： The CT flower extracts had capabilities for DPPH scavenging and high reducing power. At 100 mg/kg BW, the extract had no toxic effects on the male reproductive system. Significantly, in CT＋KET groups, CT flower extracts （50 and 100 mg/kg BW） alleviated the reduction of reproductive organ weight parameters, testosterone levels, and sperm concentration. In addition, CT flower extracts gave protection from testicular damage in KET-induced rats. Moreover, in the CT100＋KET group, CT flower extracts significantly enhanced the expression of a testicular 50-kDa tyrosine phosphorylated protein compared with that of other groups. Conclusions： C. ternatea flower extracts possessing antioxidant activity are not harmful to the male reproductive system and can protect against testicular damage in KET-induced rats.

Phenolic contents and antioxidant capacities of Thai-Makham Pom(Phyllanthus emblica L.)aqueous extracts

This study was aimed at determining the amount of the total phenolic contents （TPCs） and to evaluate the antioxidant activities of different extracts （leaves, branches, and barks） of Phyllanthus emblica （PE） L.,

Phyllanthus emblica leaf extract ameliorates testicular damage in rats with chronic stress

Stress affects the male reproductive system and can cause sub-fertility or infertility. Although Phyllanthus emblica L.(PE) extract has been shown to have high antioxidant capacity and protective properties in damaged tissue, the preventive effects of PE extract on testicular function from stress-related impairment have never been demonstrated. This study aimed to investigate the effects of PE aqueous leaf extract on testicular impairment and protein marker changes in rats suffering from chronic stress. Adult male rats were divided into four groups: a control group, a chronic stress(CS) group, and two groups with CS that received different doses of PE extract(50 or 100 mg/kg body weight(BW)). In the treatment groups, the animals were given PE extract daily before stress induction for 42 consecutive days. Stress was induced through immobilization(4 h/d) followed by forced cold swimming(15 min/d). Sperm quality and the histology of the testes and caudal epididymis were examined, as were levels of serum corticosterone, testosterone, and malondialdehyde(MDA). The expressions of testicular steroidogenic acute regulatory(StAR) and tyrosine-phosphorylated proteins were investigated using immuno-Western blot analysis, as these proteins are assumed to play important roles in spermatogenesis and androgen synthesis. The results showed that PE(50 mg/kg BW) significantly increased sperm concentration and testosterone levels, while decreasing corticosterone levels, MDA levels, sperm head abnormalities, and acrosome-reacted sperm in CS rats. In addition, PE at both doses was found to diminish testicular histopathology in the CS rats. We also found that 50 mg/kg BW of PE significantly improved StAR protein expression and altered the intensities of some tyrosine-phosphorylated proteins in testis. We conclude that PE leaf extract at 50 mg/kg BW can prevent testicular damage in rats with CS.

Evaluation of antioxidant capacity and reproductive toxicity of aqueous extract of Thai Mucuna pruriens seeds

Objective: In traditional medicine, the seeds of Thai Mucuna pruriens(T-MP) are used to treat male dysuria and are believed to enhance fertility. However, information pertaining to the toxicity of T-MP and its interaction with other properties is limited. This study was thus conducted to evaluate the antioxidant capacity and subacute toxicity of T-MP in the reproductive system.Methods: Total phenolic content and antioxidant capacity of T-MP seed extract were determined using total phenolic content, 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power assays.Male and female adult rats were treated orally with T-MP at a dosage of 150 or 300 mg/kg body weight for 14 consecutive days. Sex hormones and functional parameters in the liver and kidney were evaluated.Histopathology of all tissue was conducted using Masson’s trichrome staining. Sperm parameters, including concentration, morphology, acrosome reaction status and DNA damage, were also examined.Expression of tyrosine phosphorylated protein(Tyr Pho), androgen receptor and A-kinase-anchoring protein 4(AKAP4) were investigated using the Western blot technique.Results: T-MP seed extract contained phenolic compounds and exhibited high antioxidant capacity with no toxicity at the tested doses. It did not affect liver or kidney function parameters in the male rats, but increased estradiol, aspartate aminotransferase and alanine aminotransferase levels in the females.Additionally, it decreased serum progesterone and alkaline phosphatase levels in female rats. Serum and intratesticular testosterone levels were significantly lower in male rats that received a high dosage of T-MP. Histopathological changes were not observed in any tissue treated with T-MP. T-MP also significantly increased sperm concentration(but did not affect sperm parameters), and enhanced testicular Tyr Pho protein and androgen receptor and expression of AKAP4 in sperms.Conclusion: T-MP seed extract exhibited antioxidant capacity and was not harmful to reproductive tissues. It also had a phytoestrogenic effect on females and increased the expression of testicular and sperm markers of male fertility.

Momordica charantia fruit extract with antioxidant capacity improves the expression of tyrosine-phosphorylated proteins in epididymal fluid of chronic stress rats

Objective:Although the protective effects of Momordica charantia L.(MC)extract on chemical-induced testicular damage have been studied,the preventive effects of MC extract on functional proteins in the epididymis under chronic stress have never been reported.This study investigated the protective effects of MC fruit extract on protein secretion,especially tyrosine-phosphorylated proteins,in the epididymis of rats exposed to chronic unpredictable stress(CUS).Methods:Total phenolic compounds(TPC),total flavonoid compounds(TFC)and antioxidant capacities of MC extract were measured.Adult male rats were divided into 4 groups:control group,CUS group,and 2groups of CUS that received different doses of MC extract(40 or 80 mg/kg).In treated groups,rats were given MC daily,followed by induction of CUS(1 stressor was randomly applied from a battery of 9 potential stressors)for 60 consecutive days.Plasma corticosterone and testosterone levels were analyzed after the end of experiment.Expressions of heat-shock protein 70(HSP-70)and tyrosine-phosphorylated proteins present in the fluid of the head and tail of the epididymis were quantified using Western blot.Results:MC extract contained TPC of(19.005±0.270)mg gallic acid equivalents and TFC of(0.306±0.012)mg catechin equivalents per gram,and had 2,2-diphenyl-1-picrylhydrazyl antioxidant capacity of(4.985±0.086)mg trolox equivalents per gram,radical 50%inhibitory concentration of(2.011±0.008)mg/mL and ferric reducing antioxidant power of(23.697±0.819)μmol Fe(Ⅱ)per gram.Testosterone level in the epididymis was significantly increased,while the corticosterone level was significantly improved in groups treated with MC extract,compared to the CUS animals.Particularly,an 80 mg/kg dose of MC extract prevented the impairments of HSP-70 and tyrosine-phosphorylated protein expressions in the luminal fluid of the epididymis of CUS rats.Conclusion:MC fruit extract had antioxidant activities and improved the functional proteins secreted from the head and tail of the epididymis.It is possible to develop the MC fruit extract as a male fertility supplement for enhancing functional sperm maturation in stressed men.

Changes of testicular phosphorylated proteins in response to restraint stress in male rats

Objective： To investigate male reproductive parameters via changes of potential testicular protein markers in restraint-stress rats. Methods： Male Sprague-Dawley rats were divided into two groups （non-immobilized control and restraint-immobilized/stress groups, n=8 each group）. The stress animals were immobilized （12 h/d） by a restraint cage for 7 consecutive days. All reproductive parameters, morphology and histology were observed and compared between groups. In addition, the expression of steroidogenic acute regulatory （STAR） and phosphotyrosine proteins （previously localized in Sertoli and late spermatid cells） in testicular lysate was assayed by immuno-Western blotting. Results： Testosterone level, sperm concentration and sperm head normality of stress rats were significantly decreased while the corticosterone level was increased as compared with the control （P〈0.05）. Histologically, stress rats showed low sperm mass in epididymal lumen and some atrophy of seminiferous tubules. Although the expression of testicular STAR protein was not significantly different between groups, changed patterns of the 131, 95, and 75 kDa testicular phosphorylated proteins were observed in the stress group compared with the control group. The intensity of a tes- ticular 95-kDa phosphorylated protein was significantly decreased in stress rats. Conclusions： This study has demonstrated the alteration of testicular phosphorylated protein patterns, associated with adverse male reproductive parameters in stress rats. It could be an explanation of some infertility in stress males.